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Il ruolo del PTP in apoptosi/necrosi. Some characteristics common to several diseases: Oxidative damage Changes in Calcium homeostasis Loss of energy.

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Presentation on theme: "Il ruolo del PTP in apoptosi/necrosi. Some characteristics common to several diseases: Oxidative damage Changes in Calcium homeostasis Loss of energy."— Presentation transcript:

1 Il ruolo del PTP in apoptosi/necrosi

2 Some characteristics common to several diseases: Oxidative damage Changes in Calcium homeostasis Loss of energy production Cell death Tissue and organ dysfunction Disease

3 The Mitochondrial connection. Mitochondria are key integrators of cellular signals and stress: make life and death decisions Apoptosis and necrosis are facilitated at the level of the mitochondria by the opening of large pores (excluding extrinsic apoptotic pathway) c c c c c c mPTPBax/Bcl2 Swelling, rupture Release of proapoptotic factors IMM OMM

4 Mitochondrial Permeability transition (mPT) characterized by: Loss of inner membrane potential; Release of Cytochrome C; Cell death by apoptosis or necrosis; Cessation of mitochondrial respiration; Release of mitochondrial Ca 2+; Mitochondrial swelling (rupture) The mPT can be caused by several agents/mechanisms (e.g. ROS, Ca 2+ overload); The mPT, and as a consequence, mitochondrial dysfunction, is associated with the onset and progression of several diseases. ANT: Adenine nucleotide transporter, VDAC: voltage dependent anion channel, HK: Hexokinase; CyD: Cyclophilin D, CK: Creatine kinase, PBR: Periferal benzodiazepin receptor The Mitochondrial Permeability Transition.

5 Recenti sviluppi nella identificazione delle componenti del PTP La ATP sintetasi, maggiore produttore cellulare di ATP nella cellula, è stata recentemente proposta come lunico componente molecolare necessario per la formazione del PTP: questo dato è ancora da confermare in maniera definitiva, ma enfatizza la funzione duale dei componenti della catena respiratoria nel controllo di apoptosi e necrosi (che ritroveremo con il citocromo C)

6 Consequences of mPTP opening mitochondria swell and outer membrane rupture Ca++ and protein release (eg. Cytochrome c) mPTP opening solutes across mPTP enter mitochondria mitochondria swell and outer membrane ruptures stimulus loss of membrane potential/energy production Cell death

7 Opening of mPTP has been implicated in the aetiology and progression of several diseases including : Neurodegenerative diseases (Parkinsons, Alzheimers, MS, Huntingtons, ALS) Ischemia / reperfusion injury (AMI, Stroke, organ transplantation) Dystrophies (Bethlem, Ullrich, Duchene) Diabetic Complications (retinopathy, nephropathy) Oxidative damage, irregular calcium signalling and mitochondrial dysfunction is common to all of these diseases The mPTP and Disease.

8 Take a closer look at Ischemia / reperfusion injury (from a Mitochondrial standpoint only)

9 Heart ischemia leads to infarct size of > 70%. Reperfusion of tissue can reduce infarct size. Paradoxically reperfusion also leads to tissue death and contributes to infarct size (LRI). Methods to reduce lethal reperfusion injury should have clinical benefit Lethal reperfusion Injury.

10 PTP in Ischemia / Reperfusion Injury Whats happening in the the cell and mitochondria: Ca 2+ Calcium enters mitochondria via the Calcium Uniporter (not saturated at High [Ca 2+ ]) Ca 2+ Uniporter Ca 2+ Normal conditions Calcium is pumped out of the mitochondria via the Sodium / Calcium Antiporter (saturated at High [Ca 2+ ]) Na/Ca 2+ Antiporter Mitochondria Cell Under conditions of high cytoplasmic [Ca 2+ ] the mitochondria can overload with calcium and this can cause the mPT.

11 PTP in Ischemia / Reperfusion Injury Ischemic conditions Mitochondria Cell Loss of ATP production via respiration; increase in lactic acid and a drop in cellular pH. pH [H + ] H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ Mitochondria Cell To counter the high [H + ] the cell uses the Na + /H + Antiporter. Due to lack of ATP the Na + can not be pumped out and the cell loads with Sodium. Na + /H + Antiporter Na + H+H+ Ischemic conditions Mitochondria Cell High intracellular [Na + ] causes reversal of the Na + /Ca 2+ Antiporter and the cell loads with Ca 2+. Na + Na + /Ca 2+ Antiporter Ischemic conditions Ca 2+ Cell Restart to respiration. Large burst of ROS production. Mitochondria load with calcium via uniporter and saturate the Na + /Ca 2+ antiporter. Reoxygenation conditions Ca 2+ Ca 2+ Uniporter Na/Ca 2+ Antiporter Ca 2+ Mitochondria Cell Calcium overload and oxidative damage from ROS production induce opening of the mPTP and mitochondrial dysfunction. Cell death via necrosis/apoptosis depending on damage. Reoxygenation conditions mPTP Ca 2+ CytC Cell Death Necrosis Apoptosis Mitochondria

12 Analisi di topi KO per la ciclofilina D

13 I topi KO hanno un difetto nellapertura del PTP, ma sono normali Apertura del PTP in mitocondri isolati

14 Cellule PTP -/- sono resistenti alla morte cellulare indotta da ROS (H2O2)

15 I topi KO sono resistenti al danno cardiaco indotto da ischemia-riperfusione

16 New cardioprotective strategies. From Yellon and Hausenloy (2007 NEJM) mPTP inhibitors could be a promising strategy for lethal reperfusion injury.

17 Inibitori del PTP (CsA) sono anche efficaci nella riduzione del danno post- ischemico dellUomo Marker siericoArea infarto (NMR)

18 The mPTP is involved in the pathogenesis of several aging-associated diseases Studies using CsA and Ppif -/- mice (cyD null) have highlighted a potential role for the mPTP in the progression of several diseases. Ppif -/- mice and CsA Stroke Korde et al, 2007 Coronary Artery Bypass Graft Heart Disease Transplantation and Surgery Reperfusion Injury Alzheimers Disease ALS Neurodegenerative Disease Duchenne MD Millay et al, 2008 Dystrophies Diabetic RetinopathyHyperglycemia Doxorubicin toxicityPlatelet activation Traumatic brain injury Non-alcoholic fatty liver disease Other Myocardial infarction Piot et al 2008 Ullrich / Belthem Du et al, 2009 Keep et al, 2001 Palma et al, 2009

19 Lidentificazione di nuovi farmaci inibitori dellapertura del PTP

20 A large network of industry/academia interactions Padua: P Bernardi F Di Lisa Milan: CONGENIA PG Pelicci R Latini Nikem NMS Hamburg Oxford: EVOTEC Lyon: M Ovize

21 Ca 2+ 150 μM 1 M CsA Control Absorbance (A 540 nm) Time (mins) 0 5 10 Innovative HTS A high pulse of Ca 2+ (ca. 150 M) given to purified mitochondria will cause mPTP opening and induce mitochondrial swelling ( A 540nm ). A Ca 2+ -induced swelling assay was developed suitable for HTS Fresh Rat liver mitochondria were prepared daily and used in this assay to screen >300,000 compounds Counterscreens were run to elimate compounds that interfere with mitochondrial respiration. Several chemical classes were identified Organelle based Requiring the use of animals->liver mitochondria unprecedented at this scale

22 Adaptation of mitochondrial assays to 1536 well format Parameters to optimize: density / fitness of mitochondria concentration of stimulus (Ca ++ ) assay sensitivity (CysA) reagents volumes / order of addition reagents incubation times reagents dispensing devices readout kinetics / stability of assay signal DMSO-sensitivity reader settings (i.e. OD filter sets) Results of time-course experiment in 1536 well plates: 4 Fitzones Z: 0,74 / 0,76 / 0,70 / 0,61 Mitochondria: 1 mg/ml Calcium:300 µM Incubation:10 min Cyclosporin A:1 µM

23 HTS-Hardware in Operations 2 EVOscreen ® Mark II: biochemical assays at 1 µL level 1 EVOscreen ® Mark III: biochemical & cellular assays at 1-10 µL level

24 Class 6 compounds inhibit the mPT induced by: - Calcium overload - Oxidative damage - Protein crosslinkers - Respiration uncouplers 300μM Diamide, 40μM Ca 2+, pH 7.2, 100μM Menadione 40μM Ca 2+, pH 7.4, 50nM FCCP 40μM Ca 2+, pH 7.4, A 540 nm Time (mins) Treated GNX-A Treated GNX-A Assay performed on prepared mouse hepatic mitochondria Absorbance (A 540 nm) Time (mins) Ca 2+ 150μM, pH 7.4 Treat ed GNX- A Untreat ed A 540 nm Time (mins) Treated GNX-A A 540 nm Time (mins) Untreated PTPi block mPTP opening by several stimuli

25 State 3 inhibition (with ADP) [GNX-C] ( M) % max inhibition State 4 inhibition [GNX-C] ( M) % max inhibition 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 0510152025 -10.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 0123456789101112131415161718192021 State 3 inhibition (with ADP) [GNX-B] ( M) % max inhibition State 4 inhibition [GNX-B] ( M) % max inhibition PTPi do not have significant effects on mitochondrial respiration

26 The Calcium Retention Capacity (CRC) of purified mouse liver mitochondria is determined by measuring the point at which pulse-loaded calcium is released from the mitochondria. After the addition of a pulse of calcium (10 M) the extramitochondrial fluorescence increases. As the mitochondria take-up the calcium the fluorescence signal decreases. Calcium is continually loaded into the mitochondria until there is a sudden, large, increase in calcium fluorescence which indicates complete release of the stored calcium due to opening of the mPTP. Inhibition of the mPTP, with Cyclosporin A or our proprietary inhibitors can increase the capacity of mitochondria to retain calcium and thus protect the mitochondria from calcium overload in stress situations. Extramitochondrial calcium is measured by the fluorescence of calcium green. Note, increasing the concentration of CsA does not increase the CRC due to saturation of its target (Cyclophilin D) and is one of its limitations. Control CsA (1μM) GNX-B (0.1 M) Ca 2+ Pulses (10 M) Extra-mitochondrial Calcium fluorescence Ca 2+ Release mPTP open GNX-B (1.0 M)GNX-B (5.0 M) 40 M120 M250 M180 M380 M Time (min) CRC ( M Ca 2+ ) GNX C 0.1 M 0.5 M 1.0 M 5.0 M 0.1 M 0.5 M 1.0 M 5.0 M 0 50 100 150 200 250 300 350 CsA Control The identified PTP inhibitors are >> than CsA Class 6 compounds are able to increase the CRC (by mPTP inhibition) several fold over that of unprotected mitochondria. Class 6 compounds increase the CRC significantly over that maximally attainable with CsA.

27 PTP inhibitors for Acute Myocardial Infarction The highway to proof-of-concept: unmet medical need strong evidence for involvment of the mPTP linear path for the design of a clinical study

28 Area at Risk (g) Arae Necrotic (g) vehicle A-B GNX -B CsA 0.0 0.1 0.2 0.3 0.4 0.5 0.6 No protection Protection..................... PTP inhibitors are cardioprotective in animal models New Zealand White Rabbits were subjected to Left Anterior Descending (LAD) coronary artery occlusion for 30 mins followed by 4 hrs of reperfusion. Area at Risk and infarcted area (area necrotic) was determined by Evans blue and TTC staining. GNX-B (15 mg /kg in 3 mls 40% PEG 400; 20% DMSO) and CsA (10 mg/kg in sandimune) were administered by i.v. bolus 5 mins prior to reperfusion. Note, as with CsA in the clinical proof of concept trial, there is greater protection when the area at risk is larger. (In collaboration with Prof Ovize, Ospice di Lyon)

29 Un ruolo inaspettato per p53 nellinduzione di necrosi Dopo stress, p53 si trova localizzata insieme con la ciclofilina-D ai mitocondri

30 P53 è richiesta per lapertura del PTP durante la necrosi da stress ossidativo Viene misurata lapertura del PTP

31 Lo stress ossidativo induce necrosi e non apoptosi che dipende da p53

32 Considerazioni P53 è un regolatore fondamentale dellapoptosi, ma sembra giocare un ruolo altrettanto rilevante nella morte cellulare da necrosi dopo stress ossidativo – P53 è good e bad: in questo caso potrebbe essere coinvolto nella patogenesi di molte malattie Il coinvolgimento di p53 è unaltra dimostrazione che anche la necrosi è sottoposta (almeno in alcuni casi) a un livello di regolazione impensabile fino a poco tempo fa

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